Pure populations of astrocyte restricted precursor cells and methods for isolation and use thereof

ABSTRACT

An isolated, pure homogeneous population of mammalian astrocyte restricted precursor cells which is CD44 immunoreactive and which generate astrocytes but not oligodendrocytes is provided. Methods for identifying, isolating and using these mammalian astrocyte restricted precursor cells are also provided.

This patent application is a continuation of U.S. application Ser. No.14/202,121, filed Mar. 10, 2014, which is a continuation of U.S.application Ser. No. 12/433,060, filed Apr. 30, 2009, now issued as U.S.Pat. No. 8,673,292, which is a continuation of U.S. application Ser. No.10/502,224, filed May 17, 2005, now abandoned, which is the U.S.National Stage of PCT/US03/02356, filed Jan. 23, 2003, which claims thebenefit of priority from U.S. Provisional Application Ser. No.60/351,036, filed Jan. 23, 2002, which are herein incorporated byreference in their entirety.

This invention was supported in part by funds provided by the NationalInstitutes of Health (Grant No. 5R29NS35087-05). The U.S. Government hascertain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to a homogeneous, pure population ofmammalian astrocyte restricted precursor cells which are CD44immunoreactive and generate astrocytes but not oligodendrocytes. Thepresent invention is also related to methods for identifying andisolating a homogeneous, pure population of these mammalian astrocyterestricted precursor cells. In addition, the present invention relatesto use of mammalian astrocyte restricted precursor cells in thedevelopment of new transplantation techniques and to enhance myelinationand/or reduce necrosis and glial scar formation upon administration toanimals. The astrocyte restricted precursor cells and pharmaceuticalcompositions comprising the same, may thus be used to treat disorders ofthe nervous system resulting from trauma or disease which have in someway damaged the nerve tissue. These cells are also useful in identifyingmammalian genes specific to selected stages of development.

BACKGROUND OF THE INVENTION

Neural development has been well characterized in rodents. Multipotentcells which are nestin immunoreactive and capable of differentiatinginto astrocytes, neurons, and oligodendrocytes have been identified bymultiple investigators at various stages of development. In addition tomultipotent precursors, other more restricted precursors have also beenidentified. Different populations of cells can be distinguished bydifferences in culture conditions, self-renewal capability, as well asin their ability to integrate and to differentiate followingtransplantation.

Similar studies using human tissue are indicative of the existence ofmultiple types of neural precursors as well. Multipotent human neuralstem cells (hNSCs) have been isolated from fetal and adult tissue(Chalmers-Redman et al. Neuroscience 1997 76:1121-1128; Svendsen et al.J. Neuroscience Methods 1998 85:141-152; Vescovi et al. Exp. Neurology1999 156:71-83; Carpenter et al. Exp. Neurology 1999 158:265-278; Quinnet al. J. Neuroscience Res. 1999 57:590-602; Piper et al. J.Neurophysiology 2000 84:534-548). These cells give rise to glia andneurons, can be grown under different culture conditions, and showdifferent growth factor requirements.

Human neuron restricted precursors have also been described (Piper etal. J. Neurophysiol. 2000 84:534-548). Piper et al. used E-NCAMimmunoreactivity to isolate neuronal precursor cells while Goldman andcolleagues used neuron specific promoters to isolate neuronal precursors(Roy et al. Nat. Med. 2000 6(3):271-7; Roy et al. J. Neurosci. Res. 200059(3):321-31; Wang et al. Dev. Neurosci. 2000 22(1-2):167-76). Humanneuronal restricted precursor cells have been isolated from the adultventricular zone and hippocampus as well as from fetal tissue atmultiple stages of development. These cells differ from humanneuroepithelial cells by their expression of early neuronal markers suchas NCAM, alpha-1 tubulin and beta-III tubulin.

Proliferative adult human oligodendrocyte precursors have been isolatedfrom adult human white matter (Prabhakar et al. Brain Res. 1995672(1-2):159-69, Raine et al. Lab. Invest. 1981 45(6):534-46; Scoldinget al. Neuroreport 1995 6(3):441-5; Scolding et al. Neuroscience 199989(1):1-4) using cell surface markers. Others have usedpromoter-reporter constructs to isolate oligodendrocytes and theirprecursors from fetal and adult tissue. A2B5 immunoreactivity has beenutilized to isolate glial precursors that are capable of differentiatinginto astrocytes and oligodendrocytes (U.S. Pat. No. 6,235,527).

Quinn and colleagues (J. Neurosci. Res. 1999 57:590-602) describe amixed population of multipotent stem cells that can become altered intheir properties after prolonged culture. These cells have beensuggested to be astrocyte restricted precursor cells. However,oligodendrocyte differentiation has not been tested. Further, noinformation on antigenic expression, cytokine dependence, response togrowth factors, expression of GFAP/S100, or A2B5 is available. The cellsof Quinn et al. were obtained by sequentially passaging multipotent stemcells from cultured human spinal cord tissue.

A putative astrocyte precursor cell has also been described by Barres etal. (J. Neurosci. 1999 19(3):1049-61). This cell was isolated from theoptic nerve and its existence in any other part of the brain is unknown.This cell is A2B5 immunoreactive and thus resembles the oligodendrocyteprecursor O2A. The cells can be distinguished from the O2A cells mainlyby their failure to develop into oligodendrocytes under conditions inwhich the O2A cells readily generate oligodendrocytes. This cell isPax-6-positive and dies when exposed to serum. Immunoreactivity withCD44 is unknown.

Siedman et al. (Brain Res. 1997 753(1):18-26) have also described anastrocyte cell line derived by immortalization of a glial precursorcell. Little information on this immortalized precursor cell isavailable and its antigenic characteristics and ability to differentiateinto neurons have not been disclosed.

SUMMARY OF THE INVENTION

The present invention is directed to astrocyte restricted precursorcells, pharmaceutical compositions comprising the same, and methods ofutilizing the astrocyte restricted precursor cells to treat mammals withdamage to the nervous systems. The astrocyte restricted precursor cellsof the present invention are not immortalized. These cells do notexpress A2B5. Further, these cells differ from stem and progenitor cellpopulations in their expression of CD44 and their ability todifferentiate into astrocytes under conditions in which otherpopulations differentiate into neurons or oligodendrocytes.

Thus, one aspect of the present invention relates to an isolated, purehomogeneous population of mammalian astrocyte restricted precursor cellswhich is CD44 immunoreactive and can generate astrocytes but notoligodendrocytes.

Another aspect of the present invention relates to a method forisolating a pure homogeneous population of mammalian astrocyterestricted precursor cells. In the method of the present invention, thepure homogeneous population of astrocyte restricted precursor cells isisolated from a heterogeneous or mixed population of mammalian cells viaCD44 immunoreactivity.

Another aspect of the present invention relates to methods fordevelopment of new transplantation techniques using these mammalianastrocyte restricted precursor cells.

Another aspect of the present invention relates to pharmaceuticalcompositions comprising the mammalian astrocyte restricted precursorcells and methods of using these compositions to treat patients withdamage of the nervous system. In one embodiment, the compositions andmethods are used to enhance myelination of mammalian neuronal cells. Inanother embodiment, the compositions and methods are used to reduceglial scar formation and necrosis.

Another aspect of the present invention relates to methods foridentifying mammalian genes specific to selected stages of developmentusing these astrocyte restricted precursor cells.

DETAILED DESCRIPTION OF THE INVENTION

For cell replacement in the nervous system, differentiated cells areultimately required. However, extensive studies have shown thatdifferentiated cells do not survive well following transplantation.Therefore, some researchers have focused their efforts on use ofprecursor cells which have been shown to survive and integrate into theintact or damaged brain.

The present invention relates to a pure, homogeneous population ofmammalian astrocyte restricted precursor cells which can be isolatedfrom various sources of mammalian neural tissue and/or cells including,but not limited to, mammalian embryonic or fetal tissue, mammalianembryonic stem (ES) cell cultures, and glial restricted precursor cells.The present invention also relates to methods for isolating a pure,homogeneous population of astrocyte restricted precursor cells from suchtissues and cells.

For purposes of the present invention, by “pure” it is meant apopulation of cells in which greater than 95%, more preferably 99%,exhibit the same characteristics.

In a preferred embodiment, the mammalian tissue or cells from which theastrocyte restricted precursor cells are isolated is either rodent orhuman. However, as will be understood by those of skill in the art uponreading this disclosure, the methods for isolation taught herein arealso routinely adaptable to cells or tissues from other mammalsincluding, but not limited to, non-human primates, equines, canines,felines, bovines, porcines, ovines, lagomorphs, and the like

As demonstrated herein, the astrocyte restricted precursor cells of thepresent invention express CD44. Prior to differentiations these cellsalso express nestin. Unlike the putative astrocyte restricted cells ofBarres et al. (J. Neurosci 1999 19(3):1049-61), the astrocyte restrictedprecursor cells of the present invention do not express A2B5. Nor do thecells of the present invention express PSANCAM. The cells of the presentinvention grow well in FGF and EGF. The CD44-positive cells of thepresent invention do not express GFAP, vimentin or S-100 initially, buthave the capacity to differentiate into GFAP, vimentin and/or S-100positive cells. Upon differentiation, the cells of the present inventionmaintain their CD44 immunoreactivity but lose expression of nestin.Thus, the CD44 positive cells of the present invention can be readilydistinguished from glial-restricted precursor cells, multipotent stemcells, neuronal precursors and the putative astrocyte precursordescribed by Barres et al. (J. Neurosci. 1999 19(3):1049-61) based onantigen expression, cytokine dependence and differentiation ability. SeeTable 1 which provides a comparison of characteristics of the cells ofBarres et al with the astrocyte restricted precursor (ARP) cells of thepresent invention.

TABLE 1 ARP Cells of Characteristic Barres et al. Present Invention A2B5Expression ++ −− CD44 Expression n.d. ++ Pax-6 Expression ++ −− Clonalanalysis n.d. ++ Transplant n.d. ++ experiments Serum Exposure deathdifferentiation n.d. = not determinedThe astrocyte restricted precursor cells are present in the developingmammalian brain prior to acquisition of GFAP immunoreactivity. Inaddition, the CD44+ astrocyte restricted precursor cells can begenerated from glial-restricted precursors (GRP) and can bedistinguished from GRP cells by antigen expression, cytokine dependencyand differentiation ability.

Clonal analysis indicates that a subset of nestin+cells that are GFAP−when grown in culture differentiate solely into astrocytes. This subsetis quite large and constitutes approximately 11% of the cells analyzed.

A variety of markers were examined to identify a cell surface markerthat would label this nestin+/GFAP− population of cells. It was foundthat CD44 is specific for this glial population. CD44 expressionco-localized with astrocytic markers such as GFAP and S-100. CD44+ cellswere RC1 negative and did not co-express A2B5. A small subset of theCD44+ cells were nestin immunoreactive but GFAP negative, thusindicating that these cells represented an astrocyte precursor cellpopulation.

While the number of CD44 positive cells is small, generally in the rangeof 1-10% of the total number of cells present at any stage ofdevelopment from E15 to adult, it increases after culture in conditionsthat promote astrocyte differentiation. CD44 positive cells divide inculture and express low levels of GFAP. Expression of GFAP increasesafter differentiation while the expression of CD44 is down regulated.CD44 positive cells do not express A2B5 or PLP and, thus, can bedistinguished from the bipotential glial-restricted precursor cell.While CD44 expression has been described in other cell types such asmacrophages and astrocytes following injury, under the differentiationconditions used herein, CD44 expression was limited to astrocytes and,thus, can be used in accordance with procedures taught herein toidentify astrocyte restricted precursor cells.

Thus, as demonstrated herein, CD44 expression can be used to identifyand isolate astrocyte restricted precursor cells from various sources ofneural tissue including, but not limited to, mammalian ES cell culturesand mammalian fetal or embryonic tissue as well as glial-restrictedprecursor cells or GRPS methods for isolating glial restricted precursorcells are described in U.S. Pat. No. 6,235,527 the teachings of whichare herein incorporated by reference in their entirety. This populationof astrocyte restricted precursor cells is not immortalized. Further,population of cells does not express A2B5 and differs from stem andprogenitor populations in its expression of CD44 and its ability todifferentiate into astrocytes under conditions in which otherpopulations differentiate into neurons or oligodendrocytes.

Various methods for isolating the CD44 positive astrocyte restrictedprecursor cells from mixed populations of cells can be used.

In one embodiment, mammalian neural tubes are dissociated at a stageafter astrocyte development, for example week 10 onward in humans orafter E16 in rodents, and dissociated cells are triturated to a singlecell suspension and labeled with an anti-CD44 antibody. Labeled cellsare visualized using a fluorescently labeled secondary antibody targetedto the first antibody and labeled cells are isolated using a selectionprocess.

Examples of selection processes useful in the present invention include,but are not limited to immunopanning, magnetic bead sorting and/or FACSsorting. Detailed magnetic bead and FACS sorting protocols are wellknown in the art and can be routinely adapted to use of CD44 as theselection marker. Further, as will be understood by those of skill inthe art upon reading this disclosure, negative as well as positiveselection methods can be used. Thus enrichment of the astrocyterestricted precursor cells of the present invention can be achieved byreselecting from a mixed population cells that express CD44 but do notexpress A2B5 or E-NCAM and vice versa. Positive and negative selectionprocesses can be used in any sequence and antibodies with a bindingprofile similar to A2B5 or E-NCAM can be used.

In another embodiment, neural tubes are dissociated at any stage afterneural tube closure, for example E8.5 in mouse, E10.5 in rat, and week 5gestation in human, and cells are maintained in adherent culture for 5to 40 days. Cells are then removed from culture and CD44 positive cellsare isolated via a selection process as described in the precedingparagraph.

In another embodiment, A2B5+ cells are isolated. Cells are then inducedto differentiate in culture by growth in astrocyte promoting conditions.By astrocyte promoting conditions it is meant to include, but is notlimited to, addition of bone morphogenetic proteins (BMPs), oncostatinM, serum, Leukemia Inhibitory Factor/Ciliary Neurotrophic Factor(LIF/CTNF) and other members of the cytokine family such asinterleukin-6 either singly or in combination for a minimum period ofthree days. In a preferred embodiment these agents are added atconcentrations in the range of 1-5 ng/mL. CD44+ cells are then isolatedvia a selection process as described above.

Recently, human fetal tissue derived neural cells have become availablethrough commercial sources such as Cambrex (East Rutherford, N.J.),Clonexpress (Gaithersberg, Md.), ScienCell Research Laboratories (SanDiego, Calif.) and Clonetics (San Diego, Calif.). These cells serve as asource of neural tissue and/or cells for isolation of the mammalianastrocyte restricted precursor cells of the present invention inaccordance with the methods taught herein.

Use of human ES cell lines as a source of the astrocyte restrictedprecursor cells was also demonstrated in three human cell lines (H1, H7,H9). Human ES cells have been previously shown to differentiate intoneuronal progenitors that subsequently generate differentiated neurons(Carpenter et al. Exp. Neurol. 2001 172(2):383-97). In the presentinvention, dividing precursor cells that expressed neuronal or glialmarkers were first identified in ES cells. Differentiation conditionswere similar to those described herein and used for generating neurons.Specifically, the first stage of differentiation of the ES cells wasinduced by the formation of embryonic bodies (EBs) in FBS media with orwithout 10 μM all trans-RA. After 4 days in suspension, EBs were platedonto fibronectin coated plates in defined proliferation mediasupplemented with 10 ng/mL hEGF, 10 ng/mL hbFGF, 1 ng/mL hPDGF-AA, and 1ng/mL hIGF-1. In these conditions, the EBs adhered to the plates andcells began to migrate and proliferate on the plastic, forming amonolayer. After 3 days in these conditions many cells with neuronalmorphology were present. Similar results were found with each human EScell line.

Multiple types of dividing cell populations can be identified incultures of differentiating ES cells based on antibodies that recognizecell surface epitopes. These include A2B5+ cells, PSANCAM+ cells andCD44+ cells. Double labeling experiments following differentiationshowed that the CD44+ cells of the ES cells were a unique population ofcells that were similar morphologically, antigenically and in theirability to differentiate into astrocytes to the astrocyte restrictedcells of the present invention isolated from other sources of neuraltissue and cells.

The astrocyte restricted precursor cells of the present invention have avariety of uses.

For example, these cells can be used in nonhuman mammalian models todevelop new transplantation techniques.

In addition, these cells can be used therapeutically in mammals, morepreferably humans, in diseases characterized by neural damage and moreparticularly astrocyte degeneration. In particular, administration ofthe astrocyte restricted precursor cells of the present invention isexpected to be useful in enhancing myelination of neurons. These cellsare also useful in identifying new drugs which enhance survival andproliferation of these cells upon administration.

The cells can also be used in the reduction of scars. It is well knownthat fetal astrocytes can incorporate into the brain when transplanted.Fetal cells, as opposed to adult cells, reduce adult glial cellproliferation and scar formation, thereby promoting repair. Astrocyterestricted precursor cells of the present invention can be administeredat or near a lesioned site or area of damage one week to several weeksafter injury to reduce endogenous adult glial cell proliferation andreduce scar formation.

Accordingly, the present invention also relates to pharmaceuticalcompositions comprising these astrocyte restricted precursor cells foruse in treatment of mammals with neural damage. In a preferredembodiment, the cells are provided in injectable form or on implants topromote directed axon regeneration and reduce glial scar formation inthe forebrain, and/or in damaged spinal axons of the central nervoussystem. Such compositions are useful in promoting CNS nerve regenerationand/or enhancing myelination and/or reducing glial scar formation.Compositions comprising astrocyte restricted precursor cells can beapplied, in various different formulations as described infra, toregions or areas of nerve damage. Such compositions can be administeredto mammals having nervous system damage resulting from various causesincluding, but not limited to, trauma, surgery, ischemia, infection,metabolic disease, nutritional deficiency, malignancies andparaneoplastic syndromes, toxic agents, and degenerative disorders ofthe nervous system. Examples of neurodegenerative disorders which can betreated using compositions of the present invention include, but are notlimited to, Alzheimer's disease, Parkinson's disease, Huntington'schorea, amyotrophic lateral sclerosis, progressive supranuclear palsyand peripheral neuropathies. Compositions comprising the astrocyterestricted precursor cells of the present invention can also be appliedto a wound to reduce scar formation. For example, following surgery, acomposition comprising these cells can be applied in accordance with thepresence invention to reduce scar formation from a lesion due to, forexample, arteriovenous malformation, necrosis, bleeding, and craniotomy,which can secondarily lead to epilepsy. The compositions of the presentinvention can also be used in the treatment of epilepsy by stabilizingthe epileptic focus and reducing scar formation.

Pharmaceutical compositions of the present invention comprise aneffective amount of the isolated astrocyte restricted precursor cells ofthe present invention and a pharmaceutically acceptable carrier. By“effective amount” it is meant a composition comprising approximately100,000 to about one million cells. As will be understood by one ofskill upon reading this disclosure however, cell number may varydepending upon the selected site of administration. Examples ofpharmaceutically acceptable carriers include, but are not limited toliquid vehicles such as sterile saline, buffered saline, dextrose andwater and semi-liquid or gel-like vehicles which may further comprise amedia which impedes, at least in part, the mobility of the cells so asto localize the cells at the site of damage. Alternatively,pharmaceutically acceptable carriers may comprise a solid vehicle suchas an implant seeded or coated with the cells.

The pharmaceutical compositions can be delivered by a wide range ofmethods to promote CNS nerve regeneration, enhance myelination and/orreduce scar formation. Exemplary methods adaptable for use with thecompositions of the present invention are set forth in U.S. Pat. No.5,202,120, the teachings of which are herein incorporated by referencein their entirety.

In one embodiment, the cells are delivered by direct application, forexample, by direct injection of the cells in a vehicle into or near thesite of nerve damage. In this embodiment, it may be preferred to deliverthe cells in a vehicle comprising a media which impedes, at least inpart, the mobility of the cells so as to localize the cells at the siteof damage. Examples of media which can impede cell mobility include, butare not limited, pastes or gels, such as biodegradable gel-like polymersof fibrin or hydrogels. These semi-solid media also provide theadvantage of impeding migration of scar producing mesenchymal componentssuch as fibroblasts into the site.

In another embodiment, the cells can be delivered via a pharmaceuticalcompositions comprising a polymer implant or using surgical bypasstechniques. For example, the astrocyte restricted precursor cells can beseeded or coated onto a polymer implant. Various polymer implants withdiffering composition, geometries and pore size which can be used inthis embodiment have been described. Examples include, but are in no waylimited to, implants comprising nitrocellulose, polyanhydrides andacrylic polymers. In a preferred embodiment, an implant with a pore sizeof at least 0.45 μm is used.

The geometry of the implant is selected based upon its intended use atthe damage site. For example, an elongated triangular implant may beselected to promote nerve regeneration into the spinal cord dorsal rootentry zone while a pentagonal-shaped implant may be used to promotenerve regeneration in the corpus callosum.

In another embodiment, the polymers may serve as synthetic bridges overwhich nerve regeneration is promoted and scar formation reduced byapplication of the astrocyte restricted precursor cells at the ends orin the vicinity of the ends of the synthetic bridge. For example, anacrylic polymer tube with astrocyte restricted precursor cells of thepresent invention at one or more ends, or throughout the tube, can beused to bridge lesions rostrally or bypass lesions, for example, of thespinal cord, over which nerve regeneration can be induced. Examples ofsuch tubes are set forth in European Patent Publication 286284, and inreferences by Aebischer et al. (Brain Res. 1988 454:179-187 and Prog.Brain Res. 1988 78:599-603) and Winn et al. (Exp. Neurol. 1989105:244-250).

The cells of the present invention can also be used in combination withsurgical bypass techniques to promote nerve regeneration and/or toreduce scar formation in a selected region. Examples of such techniqueswhich can be routinely adapted to use with the compositions of thepresent invention are set forth in U.S. Pat. No. 5,202,120, which isherein incorporated by reference in its entirety.

The astrocyte restricted precursor cells of the present invention arealso useful in the identification of genes specific to selected stagesof development. In one embodiment, the cells can serve as a source ofmRNA for generation of cDNA libraries that are specific to the stagedevelopment of the cells.

The cells can also be used in the generation of cell lines andcell-specific antibodies for use therapeutically and diagnostically aswell.

The following nonlimiting examples are provided to further illustratethe present invention.

EXAMPLES Example 1 Culture of Human Neural Stem Cells

Human neural progenitor cells derived from fetal tissue were acquiredfrom Cambrex. Frozen aliquots of cells were thawed and plated onfibronectin/laminin-coated multiwell dishes in Neural Progenitor CellBasal Medium (NPBM, Cambrex) supplemented with human recombinant basicfibroblast growth factor, human recombinant epidermal growth factor,“neural survival factors”, 5 mg/mL gentamicin, and 5 mg/mLamphotericin-B (Singlequots, Cambrex). Cultures were incubated at 37°C., 5% CO₂ and fixed 24 hours later. These wells were subsequentlyprocessed for immunocytochemistry to assess the starting population ofCambrex cells. In parallel, Cambrex cells were thawed and immediatelyplated on fibronectin/laminin-coated flasks (Greiner) and cultured inNeuroepithelial Precursor (NEP) medium that consisted of DMEM-F12 (LifeTechnologies) supplemented with additives as described by Bottensteinand Sato, basic fibroblast growth factor (bFGF, 10 ng/ml, Peprotech,Rocky Hill, N.J.), and chick embryo extract (CEE, 10%). Unattached cellstypically formed floating spheres. After 24 hours in culture, sphereswere removed, gently triturated, and re-combined with the attachedcells. NEP media was exchanged every other day.

Example 2 Isolation of Human Neuroepithelial Precursor Cells (hNEPs)

After 5 days in culture, immunopanning and flow-activated cell sortingwere used to remove ENCAM+, NG2+, and A2B5+ cells. Briefly, cells weretreated with 5 mM EDTA (Life Technologies) and the suspension plated onan ENCAM antibody (5A5, Developmental Studies Hybridoma Bank)-coateddish to allow binding of all ENCAM+ cells to the plate. ENCAMantibody-coated dishes were prepared by sequentially coating tissueculture dishes with an unlabeled anti-mouse IgM antibody (10 mg/ml)overnight, rinsing dishes with DPBS, followed by coating with 5A5hybridoma supernatant for 1-3 hours at 37° C. Plates were washed twicewith DPBS prior to plating neural progenitor cells. After a 30 minuteexposure period, unbound cells (eNCAM− cells) were removed and platedonto a dish coated with antibodies to NG2 for 30 minutes. NG2 panningdishes were made by coating dishes with an NG2 antibody (1:100) for 1-3hours at 37° C. The supernatant was then removed (ENCAM−/NG2− cells) andimmunostained for A2B5. Cells were exposed to antibodies to A2B5 (1:2,Developmental Studies Hybridoma Bank) in NEP media for 1 hour at 37° C.,5% CO₂. A secondary goat anti-mouse IgM-PE labeled antibody was thenapplied for 1 hour to stain the membranes of live A2B5+ cells. All cellswere then sent through a flow-activated cell sorter to remove thepopulation of A2B5+ cells. After sorting, the negative population (humanNEPs) was propagated in NEP media on fibronectin/laminin coated T-75flasks prior to transplantation studies. NEP media was exchanged everyother day.

Example 3 Generation of Neurons, Oligodendrocytes, and Astrocytes fromhNEPs

Panned/sorted populations of human NEPs were plated onfibronectin/laminin-coated 12 mm coverslips in various conditions topromote differentiation. To induce neuronal differentiation, cells wereexposed to bFGF (10 ng/ml) and NT3 (10 ng/ml, Peprotech). After 5 daysin culture, fixed cultures were stained using antibodies to beta-IIItubulin to assess the capacity of these cells to differentiate intoneurons. For oligodendrocyte differentiation, cells were plated in abFGF (10 ng/ml)-containing medium for 2 days and then were switched to amedium containing PDGF (10 ng/ml, Upstate Biotech., Waltham, Mass.) andT3 (50 nM) for 7 days. Antibodies to O4, GalC and MBP were used toidentify oligodendrocytes in culture. For astrocytic differentiation,cells were cultured for 5 days in the presence of fetal calf serum (10%,Life Technologies). Astrocytes were identified using antibodies to CD44,GFAP and S-100.

Example 4 Clonal Cultures and Clonal Propagation

Mixed cell cultures of human fetal cells (12-22 weeks of gestation) wereobtained from Clonetics and plated in T80 flasks in the presence of bFGFand CEE (10%). After 3 days in culture cells were labeled with A2B5 andNG-2. Immunonegative cells were collected by FACS sorting analysis andreplated into flasks in the presence of bFGF and CEE. After 24 hourscells were labeled with E-NCAM, sorted by FACS and negative cells werereplated at a clonal density in 10 cm dishes in the presence of bFGF andCEE. Control dishes were labeled after 24 hours with A2B5, E-NCAM, GFAPand NG-2. At that time point, 97% of all cells do not express any of thedifferentiation markers tested. Single cells were grown at a clonaldensity of 50-200 cells/35 mm dish). Cells were maintained in FGF andCEE for 8-10 days and then CEE was withdrawn to initiatedifferentiation. For oligodendrocyte differentiation cultures wereexposed to PDGF and thyroid hormone. After 5-7 days cultures werelabeled with antibodies against GFAP and beta-III tubulin to determinedifferentiation into astrocytes and neurons, respectively. Generation ofoligodendrocytes was assessed 7 to 15 days after the initiation ofdifferentiation. Neuronal and glial differentiation was assessed usingantibodies against GFAP, and beta-III tubulin. For oligodendrocytedifferentiation, cultures were exposed to PDGF and thyroid hormone anddifferentiation was assessed using antibodies to O4 and Gal-C.

Example 5 Generation of Neurons, Oligodendrocytes, and Astrocytes fromhNEPs

Panned/sorted populations of human NEPs were plated onfibronectin/laminin-coated 12 mm coverslips in various conditions topromote differentiation. To induce neuronal differentiation, cells wereexposed to bFGF (10 ng/ml) and NT3 (10 ng/ml, Peprotech). After 5 daysin culture, fixed cultures were stained using antibodies to beta-IIItubulin to assess the capacity of these cells to differentiate intoneurons. For oligodendrocyte differentiation, cells were plated in abFGF (10 ng/ml)-containing medium for 2 days and then were switched to amedium containing PDGF (10 ng/ml, Upstate Biotech., Waltham, Mass.) andT3 (50 nM) for 7 days. Antibodies to O4, GalC and MBP were used toidentify oligodendrocytes in culture. For astrocytic differentiation,cells were cultured for 5 days in the presence of fetal calf serum (10%,Life Technologies). Astrocytes were identified using antibodies to CD44,GFAP and S-100 (Morita et al. Dev. Neurosci. 1997 19:210-218; Gomes etal. Braz. J. Med. Biol. Res. 1999 32:619-631).

Example 6 Human ES Cell Culture

Male (H1) and female (H7 & H9) huES cell lines were maintained onMATRIGEL in MEF (primary mouse embryonic fibroblasts) conditioned medium(CM). CM was generated from huES cell media (ESM) comprised of 80%Knockout DMEM (Gibco), 20% Knockout Serum replacement (Gibco), 0.1 mMbeta-mercaptoethanol, 1 mM glutamine, 1% non-essential amino acids,supplemented with 4 ng/mL hbFGF (Gibco). Cultures were passaged byincubation in 200 units/ml collagenase IV (Gibco) for about 5-10 minutesat 37° C. and then gently dissociated into small clusters in CM. Cellswere passaged about once every week. Conditioned media was generatedfrom MEF and collected daily and used immediately for feeding HuEScultures. Before addition to the HuES cultures this conditioned mediawas supplemented with an additional 4 ng/ml of hbFGF (Gibco). Cells forgenerating CM were refed with ESM daily and used for 7-10 days.

Example 7 Differentiation of huES Cells

Embryoid bodies (EBs) were formed from undifferentiated ES culturesharvested by incubation with 200 u/mL collagenase at 37° C. for 5-10minutes. The cells were gently scraped from the dish and resuspended inultra low attachment polystyrene plates (Corning) in media composed ofKO-DMEM, 20% FBS, 1% non-essential amino acids, 1 mM glutamate and 0.1mM beta-mercaptoethanol. In some experiments, 10 μM all-trans retinoicacid was added to the EBs in suspension. After 4 days in suspension, EBswere plated onto poly-l-lysine/FN coated plates in proliferation mediacomprised of DMEM/F12 with B27 and N2 supplements (Gibco) and 10 ng/mLhEGF, 10 ng/mL hbFGF (Gibco), 1 ng/mL hPDGF-AA (R&D Systems), 1 ng/mLhIGF-1 (R&D Systems). After 3 days in these conditions, the cells wereharvested with trypsin and replated in differentiation media comprisedof Neurobasal media supplemented with B27, 10 ng/mL hNT-3(R&D Systems)and 10 ng/mL hBDNF (R&D Systems). These cultures were fed 3 times perweek and fixed after 14-21 days.

Example 8 Immunocytochemistry

Cultures were stained using antibodies against A2B5 (1:2, DevelopmentalStudies Hybridoma Bank), AC133/2 (1:100, Miltenyi Biotec, Auburn,Calif.), beta-III tubulin (1:1000, Sigma), E-NCAM (1:2, 5A5,Developmental Studies Hybridoma Bank), GFAP (1:2000, Dako, Carpinteria,Calif.), NG2 (1:100) and O4 (1:2, Developmental Studies Hybridoma Bank).Following fixation, cultures were treated with 0.5% Triton X-100 (Sigma)in PBS for 2 minutes to access intracellular antigens. Fixed coverslipsor plates were then treated with primary antibodies in a blockingsolution containing Hank's balanced salt solution and 5% calf serum for1 hour at room temperature. Following 3 washes with PBS, cultures wereincubated in the appropriate secondary antibodies (1:220) conjugated toeither Texas Red or Alexa 488 (Molecular Probes, Eugene, Oreg.) for 1hour at room temperature. AC133/2 staining required amplification with abiotinylated secondary antibody, followed by a streptavidin-alexa 488conjugated tertiary antibody. All cultures were counterstained with DAPI(Molecular Probes) to identify cell nuclei.

What is claimed is:
 1. An isolated population of mammalian precursorcells which generate astrocytes but not oligodendrocytes, said cellsbeing CD44+.
 2. A method for treating damaged neural cells in a mammal,said method comprising administering to a mammal with damaged neuralcells the mammalian precursor cells of claim
 1. 3. The method of claim 2wherein the mammalian precursor cells are administered to the mammal bydirect injection into or near a site of nerve damage.
 4. The method ofclaim 2 wherein the mammalian precursor cells are administered to themammal as an implant seeded or coated with the mammalian precursorcells.
 5. The method of claim 4 wherein the implant is implanted at ornear a site of damaged neural cells in the mammal.
 6. The method ofclaim 2 wherein administering the mammalian precursor cells to themammal enhances survival of the damaged neural cells.
 7. The method ofclaim 2 wherein administering the mammalian precursor cells to themammal reduces scar formation.
 8. An astrocyte generated ordifferentiated from a population of mammalian precursor cells which areCD44+ and which generate astrocytes but not oligodendrocytes.
 9. Anastrocyte generated or differentiated from a mammalian precursor cellwhich generate astrocytes but not oligodendrocytes, said precursor cellbeing nestin+, A2B5−, and E-NCAM− or Pax-6−.
 10. A method of identifyinga population of mammalian astrocyte restricted precursor cells whichgenerate astrocytes but not oligodendrocytes, said method comprisingidentifying from a heterogeneous or mixed population of mammalian cellsor tissue CD44+ cells that differentiate to astrocytes but notoligodendrocytes.
 11. The method of claim 10 further comprisingisolating from the heterogeneous or mixed population of mammalian cellsor tissue the CD44+ cells that differentiate to astrocytes but notoligodendrocytes.
 12. The method of claim 11 wherein the CD44+ cellsthat differentiate to astrocytes but not oligodendrocytes are isolatedby immunopanning, magnetic bead sorting and/or FACS sorting using CD44as the selection marker.
 13. The method of claim 11 wherein the isolatedpopulation of CD44+ cells that differentiate to astrocytes but notoligodendrocytes is enriched by reselecting from a mixed populationcells that express CD44 but do not express E-NCAM.
 14. The method ofclaim 11 wherein the isolated population of CD44+ cells thatdifferentiate to astrocytes but not oligodendrocytes is enriched byreselecting from a mixed population cells that express CD44 but do notexpress A2B5.
 15. The method of claim 11 wherein the isolated CD44+cells that differentiate to astrocytes but not oligodendrocytes generateastrocytes under conditions in which other cell populationsdifferentiate into neurons or oligodendrocytes.